Includes bibliographical references (leaves 63-86). / Disease resistance in plants has been extensively studied for the past century with many new and exciting results being discovered each year. A plant utilises both preformed and induced defence responses to resist pathogen attack but researchers have focused on dissecting the induced defence response pathway. The complex signal transduction pathway underlying the establishment of resistance to a wide range of pathogen attack is currently being dissected using Arabidopsis thaliana as a model organism. Arabidopsis mutants displaying altered disease resistance response to pathogen infections can help us to get a beUer understanding of the genetiC and molecular basis of the disease resistance pathway. Extensive research has shown that accumulation of 3 signalling molecules are vitally important for establishing a resistance response, as aberrant signalling or accumulation of salicylic acid , ethylene or jasmonic acid `leads to an altered resistance response. Researchers continue to isolate and characterise defence-related mutants to piece together the intricate puzzle of defence-signalling components. A dominant Arabidopsis mutant, constitutive induced resistance 3 (cir3), had been isolated from an ethylmethane sulfonate (EMS) mutagenised transgenic line expressing luciferase under the control of the PR-1 promoter (PR-1

Word processed copy. / Includes bibliographical references. / This study investigated the link between the association of the yeast linker histone homologue, Hholp, and the compaction of the yeast genome during stationary phase. The relative gene content of condensed chromatin, fractionated and isolated by sucrose gradient ultracentrifugation from stationary and exponential phase cultures was compared using genome-wide technologies. This study showed that condensed chromatin of stationary phase culture contained an enriched density of genes on all the chromosomes, indicating global compaction of the yeast genome during stationary phase.

Bibliography: leaves 90-111. / It is well established that histone H1 is involved in facilitating the higher-order structures of chromatin. It has not become possible to test the biochemical function of H1 in Saccharomyces cerevisiae, since the identification of a H1 homologue (Landsman, 1996) with properties of a bona fide linker histone (Patterton et al., 1998; Ali, 2000). Such studies were difficult since no phenotype for a HHO1 deleted strain has been published. In this study I have investigated the response of an HHO1 disrupted strain to numerous conditions including UV radiation, osmotic and oxidative stress, y-irradiation and longevity.

Includes abstract. / Includes bibliographical references. / In order to interpret the results obtained from a microarray experiment, researchers often shift focus from analysis of individual differentially expressed genes to analyses of sets of genes. These gene-set analysis (GSA) methods use previously accumulated biological knowledge from databases such as the Gene Ontology (GO) or KEGG to group genes into sets based on their annotations. They aim to rank these gene sets in a way that reflects their relative importance in the experimental situation in question. The objective is that this approach reveals sets of genes with subtle but coordinated behaviour implicating specific biological processes or pathways in the response under study. Several GSA methods have been proposed and debates have ensued on the statistical foundations of the different approaches and the various hypothesis tests used. In particular, criticism has been directed at methods that rely on a strict cut-off to determine significant genes and those that assume genes are expressed independently. We show that paralogs, which typically have high sequence identity and similar molecular functions also exhibit high correlation in their expression patterns. This, together with the fact that the calculation of gene-set significance by all GSA methods is influenced by the number of genes in the gene set, means that sets with high numbers of paralogs are ranked in a biased manner that reflects more the redundant and dependent nature of para logs than any biological phenomenon.

Includes bibliographical references. / Bacterial strains were isolated by enrichment cultures from oil-contaminated soil samples. In the present study, several strains, capable of growing on crude oil, were isolated. Isolates were screened for their inherent abilities to produce bioemulsifiers when they were grown on hydrocarbon substrates.

Includes bibliographical references. / Skin cancer is the most common cancer worldwide, and its incidence rate in South Africa is increasing. Photodynamic therapy (PDT) has been shown to be an effective treatment modality, through topical administration, for treatment of non-melanoma skin cancers. Our group investigates hypericin-induced PDT (HYP-PDT) for the treatment of both non-melanoma and melanoma skin cancers. However, a prerequisite for effective cancer treatments is efficient and selective targeting of the tumoral cells with minimal collateral damage to the surrounding normal cells, as it is well know that cancer therapies have bystander effects on normal cells in the body, often causing undesirable side effects. PDT can induce a bystander effect, defined as indirect damaged induced into adjacent cells either via intercellular gap junctions or via diffusible ROS released in the microenvironment. It is therefore important to know the effects of HYP-PDT on the normal cell population surrounding the non-melanoma skin cancer or melanoma tumor. The aim of this project was to investigate the cellular and molecular effects of HYP-PDT on normal primary human keratinocytes (Kc), melanocytes (Mc) and fibroblasts (Fb) in an in vitro tissue culture model thus representing both the epidermal and dermal cellular compartments of human skin.

Includes bibliographical references. / Marine environments were investigated and actinomycetes were isolated on selective media. Thirty-four (34) actinomycete strains were isolated and identified: 21 Micromonospora strains, 10 Streptomyces strains, and 3 Pseudonocardia strains. A polyphasic approach was employed to determine the novelty of the isolates. Potentially, all 21 Micromonospora strains are novel, as revealed by an original identification scheme developed to assess quickly and easily the novelty of newly isolated environmental Micromonospora strains. Standardized media for testing physiological characters of Micromonospora strains were developed, and additional physiological characteristics of 15 of the validly published members of the genus Micromonospora are described. Furthermore, 14 of the 15 validly published Micromonospora species, and 20 of the 21 environmental Micromonospora isolates grew under anaerobic conditions.

Includes bibliographical references (leaves 77-96). / This study focused on the potential use of E. natalensis seedlings, callus cultures and hairy root cultures as alternative sources of diospyrin. Chloroform extracts of E. natalensis seedling organs showed antimycobacterial activity against Mycobacterium aurum, and HPLC analysis of the extracts demonstrated a corresponding accumulation of diospyrin, mostly in the stem (0.23%, DW) and roots (0.17%, DW) of the seedlings.

Transforming growth factor beta-1 (TGF-β1) is a cytokine involved in immune modulation and tissue regeneration and has a polymorphic TGFB1 promoter. African-specific single nucleotide polymorphisms (SNPs) have been identified in the TGFB1 promoter and they occur at higher frequencies in South Africans with African-genetic ancestry (≈17%) compared with West and East African genomes (<5%). However, the functional significance of these SNPs has only been partially explored. In this study it was hypothesized that higher frequencies of immunemediated disease complications in South Africans, such as HIV-associated nephropathy (HIVAN), may be influenced by functional genetic variations in TGFB1. To address this, the African-specific TGFB1 haplotypes containing singular, or combinations of, -1287 G>A (rs11466314), -1154 C>T (rs35318502), -387 C>T (rs11466316) and -14 G>A (rs9282871) were investigated for their effect on TGFB1 promoter activity. Briefly, an extended TGFB1 regulatory region driving a luciferase reporter was used as a template to generate six TGFB1 promoter haplotypes (referred to as H-1 through H-6 in this thesis) by site-directed mutagenesis and luciferase activity was used to measure promoter activity. The functional TGFB1 -1347 C>T variant was also investigated (H-5 and H-6 containing -1347 C and T alleles, respectively) because all four of the African variants more frequently co-occurred with the previously reported "lower expressing" TGFB1 -1347 C variant (H-1 through H- 4). Transient transfection of the TGFB1 promoter reporter constructs in two renal cell lines (RCC4+VHL and Caki- 2) showed no difference between -1347 C (H-5) and T (H-6) basal promoter activity. Having at least one African variant resulted in ~5-fold loss of basal TGFB1 promoter activity in renal cells when compared to the most common haplotype (H-5) (p<0.05). The repressive effect is mainly attributed to the -387 T variant (H-1) as the addition of other African variants on this haplotype showed no additional TGFB1 promoter repression. The repressive effect of the TGFB1 -387 T variant was maintained even after in-vitro treatment of transfected renal cells with recombinant human TGF-β1 (rhTGF-β1). To determine whether the African-specific TGFB1 promoter haplotypes (H-AFR), containing TGFB1 -387 T and tested in luciferase assays, also impact on endogenous TGF-β1 protein levels, western blot analysis was performed on human dermal fibroblasts from patients who had been genotyped. Similar to the promoter studies, basal TGF- β1 protein levels in cells with the H-AFR were ~47% lower compared to cells without (p=0.04) and no difference was seen in response to hrTGF-β1. Interestingly, when western blots were screened for phosphorylated Smad3 (pSmad3) protein levels, as an indicator of the activated TGF-β1 canonical pathway, similar pSmad3 levels were observed under basal and hrTGF-β1 stimulated conditions for all haplotypes (p>0.05). The possible interaction of HIV tat on the African TGFB1 promoter variants was also assessed. Luciferase activity was measured after co-transfecting renal RCC4+VHL and HT1080 fibroblast cells with a HIV Tat expression vector and the H-6, H-5 and H-AFR promoter luciferase constructs. Results showed that the promoter activity for all TGFB1 haplotypes was upregulated in the renal cells (≥1.6-fold; p<0.001). The same result was, however, only observed for TGFB1 haplotypes H-5 and H-AFR in the fibroblast cells (≥1.4-fold; p<0.01). This is interesting because no difference was seen between TGFB1 H-5 and H-6 basal promoter activity. To investigate whether histopathological severity of HIVAN correlated with TGF-β1 staining patterns, immunohistochemistry was performed on 20 renal biopsies from patients with HIVAN. A semi-quantitative "histo" score (H-score) was calculated by multiplying the percentage of positive cells with the intensity of the stain before comparing the scores with control biopsies (HIV-negative, n=3 and HIV-positive without HIVAN, n=3). Compared to the HIV-positive controls the kidney tubules of HIVAN biopsies had higher H-scores. Strikingly, the interstitium of HIVAN samples stained much more prominently (17/18) than both control groups suggesting that TGF-β1 staining in the renal interstitium appear to be specific for HIVAN. In conclusion this study shows that the African-specific haplotypes effect basal TGFB1 promoter activity and TGF-β1 protein levels. However, they do not seem to affect the cytoplasmic TGF-β1/pSmad3 protein levels in response to rhTGF-β1 autocrine stimulation. Immunohistochemistry results suggest that TGF-β1 pathway may be prominently dysregulated in the renal interstitium of HIVAN cases.

Second degree burns result in the destruction of the skin as well as its adnexa. Following such burns the wound heals without the formation of skin appendages and hair follicles. In normal embryonic development hair follicle formation requires the interaction between epithelial (keratinocytes) and mesenchyme cells. Attempts to recapitulate the process of hair induction in wounded skin in vitrousing human cells have to date been unsuccessful. The aim of this project is to attempt to elicit the early steps of hair follicle formation (induction) by co-culturing primary human keratinocytes with embryonic murine mesenchyme cells and assessing changes in expression patterns of genes associated with or reflective of induction. Mesenchymal cells and keratinocytes cells were obtained by enzymatically digesting dorsal neonatal mouse skin and neonatal human foreskin using dispase and collagenase. Cells were cultured separately, and their growth dynamics measured. The isolated neonatal mouse mesenchymal cells were shown to have hair induction potential because they expressed dermal papilla signature genes, Alp, Sox2 and Vcan. However, this characteristic was lost during in vitro propagation suggesting that mesenchymal cells lose their hair inductive potential during culture. In contrast, when cultured at high densities or in spheroids in hanging drops, the dermal papilla signature genes were upregulated suggesting that this might be a way to maintain inductive potential. Primary foreskin keratinocytes expressed high levels of basal layer marker, keratin 5 (K5), and low levels of the early differentiation marker, K10, suggesting that the isolated keratinocytes have stem cell properties. When co-cultured with neonatal mouse mesenchymal cells using Transwells, the mesenchymal cells were able to elicit colony formation on keratinocytes in co-cultures, indicating that they support keratinocyte proliferation. It was not possible to do hair follicle induction marker analysis of human foreskin keratinocytes cocultures because of challenges and difficulties encountered during expansion. Therefore, Immortalised HaCaT keratinocytes were tested. HaCaT keratinocytes were shown to be induced during cocultures because they upregulated Wnt signalling genes, β-catenin and NF-KB. As an additional approach, human foreskin keratinocytes were cultured in medium containing Wnt signalling pathway ligand, Wnt3a. β-catenin and NF-KB were slightly reduced, and only Lef1 was upregulated in human foreskin keratinocytes cultured in Wnt3a conditioned medium. The results of this study show that neonatal mouse mesenchymal cells have hair inducing capabilities and it is lost by in vitro propagation and can be restored by spheroid cell culture. The results also demonstrate that human foreskin keratinocytes need to be expanded using efficient culture methods to maintain an undifferentiated state.